Complexity Management of Genomic DNA

ABSTRACT

The presently claimed invention provides for novel methods and kits for analyzing a collection of target sequences in a nucleic acid sample. A sample is amplified under conditions that enrich for a subset of fragments that includes a collection of target sequences. The invention further provides for analysis of the above sample by hybridization to an array, which may be specifically designed to interrogate the collection of target sequences for particular characteristics, such as, for example, the presence or absence of one or more polymorphisms.

RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. applicationSer. No. 09/916,135 filed Jul. 25, 2001 the disclosure of which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to enrichment and amplification of sequences froma nucleic acid sample. In one embodiment, the invention relates toenrichment and amplification of nucleic acids for the purpose of furtheranalysis. The present invention relates to the fields of molecularbiology and genetics.

BACKGROUND OF THE INVENTION

The past years have seen a dynamic change in the ability of science tocomprehend vast amounts of data. Pioneering technologies such as nucleicacid arrays allow scientists to delve into the world of genetics in fargreater detail than ever before.

Exploration of genomic DNA has long been a dream of the scientificcommunity. Held within the complex structures of genomic DNA lies thepotential to identify, diagnose, or treat diseases like cancer,Alzheimer disease or alcoholism. Exploitation of genomic informationfrom plants and animals may also provide answers to the world's fooddistribution problems.

Recent efforts in the scientific community, such as the publication ofthe draft sequence of the human genome in February 2001, have changedthe dream of genome exploration into a reality. Genome-wide assays,however, must contend with the complexity of genomes; the human genomefor example is estimated to have a complexity of 3×10⁹ base pairs. Novelmethods of sample preparation and sample analysis that reduce complexitymay provide for the fast and cost effective exploration of complexsamples of nucleic acids, particularly genomic DNA.

Single nucleotide polymorphisms (SNPs) have emerged as the marker ofchoice for genome wide association studies and genetic linkage studies.Building SNP maps of the genome will provide the framework for newstudies to identify the underlying genetic basis of complex diseasessuch as cancer, mental illness and diabetes. Due to the wide rangingapplications of SNPs there is still a need for the development ofrobust, flexible, cost-effective technology platforms that allow forscoring genotypes in large numbers of samples.

SUMMARY OF THE INVENTION

In one embodiment a method of reducing the complexity of a first nucleicacid sample to produce a second nucleic acid sample is disclosed. Themethod comprises first selecting a collection of target sequences by amethod comprising: identifying fragments that are in a selected sizerange when a genome is digested with a selected enzyme or enzymecombination; identifying sequences of interest present on the fragmentsin the selected size range; and selecting as target sequences fragmentsthat are in the selected size range and comprise a sequence of interest.The first nucleic acid sample is fragmented to produce sample fragmentsand at least one adaptor is ligated to the sample fragments. A secondnucleic acid sample is generated by amplifying the sample. The amplifiedsample is enriched for a subset of the sample fragments and that subsetincluded a collection of target sequences. In one embodiment the subsetof sample fragments is targeted for enrichment by selecting the methodof fragmentation.

In one embodiment amplification of the sample is by PCR using 20 to 50cycles. A single primer complementary to the adaptor may be used in someembodiments. In some embodiments two different adaptors are ligated tothe fragments and two different primers are used for amplification. Inyet another embodiment a single adaptor is used but the adaptor has adouble stranded region and single stranded regions. Primers to thesingle stranded regions are used for amplification. In one embodimentthe adaptor sequence comprises a priming site. In another embodiment theadaptor comprises a tag sequence.

In one embodiment the step of fragmenting the first nucleic acid sampleis by digestion with at least one restriction enzyme. The restrictionenzyme may, for example, have a 6 base recognition sequence or an 8 baserecognition sequence. In some embodiments a type IIs endonuclease isused. In one embodiment fragmenting, ligating and amplifying steps aredone in a single tube.

In one embodiment the second nucleic acid sample comprises at least0.01%, 0.5%, 3%, 12% or 50% of the first nucleic acid sample. The firstnucleic acid sample may be, for example, genomic DNA, DNA, cDNA derivedfrom RNA or cDNA derived from mRNA.

In one embodiment the target sequences are 800, 1000, 1200, 1500, or2000 base pairs long or less. In one embodiment the subset of samplefragments enriched in the second nucleic acid sample is comprised offragments that are primarily 2000 or 3000 base pairs long or less.

In one embodiment target sequences contain one or more sequence ofinterest, such as, for example, sequence variations, such as SNPs. Insome embodiments a SNP may be associated with a phenotype, a disease,the efficacy of a drug or with a haplotype.

In another embodiment a method for selecting a collection of targetsequences is disclosed. The steps of the method are identifyingfragments that are in a selected size range when a genome is digestedwith a selected enzyme or enzyme combination; identifying sequences ofinterest present on the fragments in the selected size range; andselecting as target sequences fragments that are in the selected sizerange and comprise a sequence of interest. In one embodiment a computersystem is used for one or more steps of the method. In one embodiment anarray is designed to interrogate one or more specific collections oftarget sequences. In another embodiment a collection of target sequencesis disclosed. The collection may be amplified. The collection may alsobe attached to a solid support.

In another embodiment a method is disclosed for analyzing a collectionof target sequences by providing a nucleic acid array; hybridizing theamplified collection of target sequences to the array; generating ahybridization pattern resulting from the hybridization; and analyzingthe hybridization pattern. In one embodiment the array is designed tointerrogate sequences in the collection of target sequences. In oneembodiment the sequences are analyzed to determine if they containsequence variation, such as SNPs.

In another embodiment a method for genotyping an individual isdisclosed. A collection of target sequences comprising a collection ofSNPs is amplified and hybridized to an array comprising probes tointerrogate for the presence or absence of different alleles in thecollection of SNPs. The hybridization pattern is analyzed to determinewhich alleles are present for at least one of the SNPs.

In another embodiment a method for screening for DNA sequence variationsin a population of individuals is disclosed. Amplified target sequencesfrom each individual are hybridized to an array that interrogates forsequence variation. The hybridization patterns from the arrays arecompared to determine the presence or absence of sequence variation inthe population of individuals.

In another embodiment kits for genotyping individuals or samples aredisclosed. The kit may contain one or more of the following components:buffer, nucleotide triphosphates, a reverse transcriptase, a nuclease,one or more restriction enzymes, two or more adaptors, a ligase, a DNApolymerase, one or more primers and instructions for the use of the kit.In one embodiment the kit contains an array designed to interrogatesequence variation in a collection of target sequences.

In another embodiment a solid support comprising a plurality of probesattached to the solid support is disclosed. The probes may be designedto interrogate sequence variation in a collection of target sequences.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a Venn diagram illustrating how a collection of targetsequences may be selected. Potential target sequences are found at theintersection between the set of fragments that contain sequences ofinterest and the fragments that are in a selected size range. Theselected size range is within the set of fragments that are efficientlyamplified by PCR under standard conditions.

FIG. 2 shows how in silico digestion can be used to predict the size ofrestriction fragments containing SNPs.

FIG. 3 is a table of the number of SNPs predicted to be found on 400 to800 base pair fragments when genomic DNA is digested with therestriction enzyme in column 1.

FIG. 4 is a flow chart showing design of an array in conjunction withsize selection of SNP containing fragments.

FIG. 5 is a schematic of a method in which the ends of the adaptors arenon-complementary and the fragments are amplified with a primer pair.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS (A) General

The present invention relies on many patents, applications and otherreferences for details known to those of the art. Therefore, when apatent, application, or other reference is cited or repeated below, itshould be understood that it is incorporated by reference in itsentirety for all purposes as well as for the proposition that isrecited. As used in the specification and claims, the singular form “a,”“an,” and “the” include plural references unless the context clearlydictates otherwise. For example, the term “an agent” includes aplurality of agents, including mixtures thereof. An individual is notlimited to a human being but may also be other organisms including butnot limited to mammals, plants, bacteria, or cells derived from any ofthe above.

Throughout this disclosure, various aspects of this invention arepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well ascommon individual numerical values within that range. For example,description of a range such as from 1 to 6 should be considered to havespecifically disclosed subranges such as from 1 to 3, from 1 to 4, from1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well asindividual numbers within that range, for example, 1, 2, 3, 4, 5, and 6.The same holds true for ranges in increments of 10⁵, 10⁴, 10³, 10², 10,10⁻¹, 10⁻², 10⁻³, 10⁻⁴, or 10⁻⁵, for example. This applies regardless ofthe breadth of the range.

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques of organic chemistry, polymertechnology, molecular biology (including recombinant techniques), cellbiology, biochemistry, and immunology, which are within the skill of theart. Such conventional techniques include polymer array synthesis,hybridization, ligation, and detection of hybridization using a label.Specific illustrations of suitable techniques can be had by reference tothe example hereinbelow. However, other equivalent conventionalprocedures can, of course, also be used. Such conventional techniquescan be found in standard laboratory manuals such as Genome Analysis: ALaboratory Manual Series (Vols. I-IV), Using Antibodies: A LaboratoryManual, Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, andMolecular Cloning: A Laboratory Manual (all from Cold Spring HarborLaboratory Press), all of which are herein incorporated in theirentirety by reference for all purposes.

Some aspects of the present invention make use of microarrays, alsocalled arrays. Methods and techniques applicable to array synthesis havebeen described in U.S. Pat. Nos. 5,143,854, 5,242,974, 5,252,743,5,324,633, 5,384,261, 5,424,186, 5,451,683, 5,482,867, 5,491,074,5,527,681, 5,550,215, 5,571,639, 5,578,832, 5,593,839, 5,599,695,5,624,711, 5,631,734, 5,795,716, 5,831,070, 5,837,832, 5,856,101,5,858,659, 5,936,324, 5,968,740, 5,974,164, 5,981,185, 5,981,956,6,025,601, 6,033,860, 6,040,193, and 6,090,555. All of the above patentsincorporated herein by reference in their entireties for all purposes.

The practice of the present invention may also employ conventionalbiology methods, software and systems. Computer software products of theinvention typically include computer readable medium havingcomputer-executable instructions for performing the logic steps of themethod of the invention. Suitable computer readable medium includefloppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM,magnetic tapes and etc. The computer executable instructions may bewritten in a suitable computer language or combination of severallanguages. Basic computational biology methods are described in, e.g.Setubal and Meidanis et al., Introduction to Computational BiologyMethods (PWS Publishing Company, Boston, 1997); Salzberg, Searles,Kasif, (Ed.), Computational Methods in Molecular Biology, (Elsevier,Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics:Application in Biological Science and Medicine (CRC Press, London, 2000)and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysisof Gene and Proteins (Wiley & Sons, Inc., 2^(nd) ed., 2001).

The present invention may also make use of various computer programproducts and software for a variety of purposes, such as probe design,management of data, analysis, and instrument operation. See, U.S. Pat.Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555,6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170.

Additionally, the present invention may have preferred embodiments thatinclude methods for providing genetic information over networks such asthe Internet as shown in U.S. patent application Ser. Nos. 10/063,559,60/349,546, 60/376,003, 60/394,574, and 60/403,381.

The word “DNA” may be used below as an example of a nucleic acid. It isunderstood that this term includes all nucleic acids, such as DNA andRNA, unless a use below requires a specific type of nucleic acid.

(B) Definitions

Nucleic acids according to the present invention may include any polymeror oligomer of pyrimidine and purine bases, preferably cytosine,thymine, and uracil, and adenine and guanine, respectively. (See AlbertL. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)which is herein incorporated in its entirety for all purposes). Indeed,the present invention contemplates any deoxyribonucleotide,ribonucleotide or peptide nucleic acid component, and any chemicalvariants thereof, such as methylated, hydroxymethylated or glucosylatedforms of these bases, and the like. The polymers or oligomers may beheterogeneous or homogeneous in composition, and may be isolated fromnaturally occurring sources or may be artificially or syntheticallyproduced. In addition, the nucleic acids may be DNA or RNA, or a mixturethereof, and may exist permanently or transitionally in single-strandedor double-stranded form, including homoduplex, heteroduplex, and hybridstates.

An “oligonucleotide” or “polynucleotide” is a nucleic acid ranging fromat least 2, preferably at least 8, 15 or 20 nucleotides in length, butmay be up to 50, 100, 1000, or 5000 nucleotides long or a compound thatspecifically hybridizes to a polynucleotide.

Polynucleotides of the present invention include sequences ofdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or mimeticsthereof which may be isolated from natural sources, recombinantlyproduced or artificially synthesized. A further example of apolynucleotide of the present invention may be a peptide nucleic acid(PNA). (See U.S. Pat. No. 6,156,501 which is hereby incorporated byreference in its entirety.) The invention also encompasses situations inwhich there is a nontraditional base pairing such as Hoogsteen basepairing which has been identified in certain tRNA molecules andpostulated to exist in a triple helix. “Polynucleotide” and“oligonucleotide” are used interchangeably in this application.

The term “fragment,” “segment,” or “DNA segment” refers to a portion ofa DNA polynucleotide, DNA or chromosome. A polynucleotide, for example,can be broken up, or fragmented into, a plurality of segments. Variousmethods of fragmenting nucleic acid are well known in the art. Thesemethods may be, for example, either chemical or physical in nature.Chemical fragmentation may include partial degradation with a DNase;partial depurination with acid; the use of restriction enzymes;intron-encoded endonucleases; DNA-based cleavage methods, such astriplex and hybrid formation methods, that rely on the specifichybridization of a nucleic acid segment to localize a cleavage agent toa specific location in the nucleic acid molecule; or other enzymes orcompounds which cleave DNA at known or unknown locations. Physicalfragmentation methods may involve subjecting the DNA to a high shearrate. High shear rates may be produced, for example, by moving DNAthrough a chamber or channel with pits or spikes, or forcing the DNAsample through a restricted size flow passage, e.g., an aperture havinga cross sectional dimension in the micron or submicron scale. Otherphysical methods include sonication and nebulization. Combinations ofphysical and chemical fragmentation methods may likewise be employedsuch as fragmentation by heat and ion-mediated hydrolysis. See forexample, Sambrook et al., “Molecular Cloning: A Laboratory Manual,”3^(rd) Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001) (“Sambrook et al.) which is incorporated herein by reference forall purposes. These methods can be optimized to digest a nucleic acidinto fragments distributed around a selected size range. Included inthis digestion will be many fragments that are shorter than the selectedrange and many fragments that are longer than the selected range butmany fragments will be within the selected size range. Useful sizeranges may be from 1, 100, 200, 400, 700, 1000 or 2000 to 500, 800,1500, 2000, 4000 or 10,000 base pairs. However, larger size ranges suchas 4000, 10,000 or 20,000 to 10,000, 20,000 or 500,000 base pairs mayalso be useful.

A number of methods disclosed herein require the use of restrictionenzymes to fragment the nucleic acid sample. In general, a restrictionenzyme recognizes a specific nucleotide sequence of four to eightnucleotides and cuts the DNA at a site within or a specific distancefrom the recognition sequence. For example, the restriction enzyme EcoRIrecognizes the sequence GAATTC and will cut a DNA molecule between the Gand the first A. The length of the recognition sequence is roughlyproportional to the frequency of occurrence of the site in the genome. Asimplistic theoretical estimate is to that a six base pair recognitionsequence will occur once in every 4096 (4⁶) base pairs while a four basepair recognition sequence will occur once every 256 (4⁴) base pairs. Insilico digestions of sequences from the Human Genome Project show thatthe actual occurrences may be even more infrequent for some enzymes andmore frequent for others, for example, PstI cuts the human genome moreoften than would be predicted by this simplistic theory while SalI andXhoI cut the human genome less frequently than predicted. Because therestriction sites are rare, the appearance of shorter restrictionfragments, for example those less than 1000 base pairs, is much lessfrequent than the appearance of longer fragments. Many differentrestriction enzymes are known and appropriate restriction enzymes can beselected for a desired result. (For a description of many restrictionenzymes see, New England BioLabs Catalog (Beverly, Mass.) which isherein incorporated by reference in its entirety for all purposes).

Information about the sequence of a region may be combined withinformation about the sequence specificity of a particular restrictionenzyme to predict the size, distribution and sequence of fragments thatwill result when a particular region of a genome is digested with thatenzyme. In silico digestion is a computer aided simulation of enzymaticdigests accomplished by searching a sequence for restriction sites. Insilico digestion provides for the use of a computer or computer systemto model enzymatic reactions in order to determine experimentalconditions before conducting any actual experiments. An example of anexperiment would be to model digestion of the human genome with specificrestriction enzymes to predict the sizes and sequences of the resultingrestriction fragments.

“Adaptor sequences” or “adaptors” are generally oligonucleotides of atleast 5, 10, or 15 bases and preferably no more than 50 or 60 bases inlength, however, they may be even longer, up to 100 or 200 bases.Adaptor sequences may be synthesized using any methods known to those ofskill in the art. For the purposes of this invention they may, asoptions, comprise templates for PCR primers, restriction sites, tags andpromoters. The adaptor may be partially, entirely or substantiallydouble stranded. The adaptor may be phosphorylated or unphosphorylatedon one or both strands. Modified nucleotides, for example,phosphorothioates, may also be incorporated into one or both strands ofan adaptor.

Adaptors are particularly useful in some embodiments of the methods ifthey comprise a substantially double stranded region and short singlestranded regions which are complementary to the single stranded regioncreated by digestion with a restriction enzyme. For example, when DNA isdigested with the restriction enzyme EcoRI the resulting double strandedfragments are flanked at either end by the single stranded overhang5′-AATT-3′, an adaptor that carries a single stranded overhang5′-AATT-3′ will hybridize to the fragment through complementaritybetween the overhanging regions. This “sticky end” hybridization of theadaptor to the fragment may facilitate ligation of the adaptor to thefragment but blunt ended ligation is also possible.

In some embodiments the same adaptor sequence is ligated to both ends ofa fragment. Digestion of a nucleic acid sample with a single enzyme maygenerate similar or identical overhanging or sticky ends on either endof the fragment. For example if a nucleic acid sample is digested withEcoRI both strands of the DNA will have at their 5′ ends a singlestranded region, or overhang, of 5′-AATT-3′. A single adaptor sequencethat has a complementary overhang of 5′-AATT-3′ can be ligated to eachend of the fragment.

A single adaptor can also be ligated to both ends of a fragmentresulting from digestion with two different enzymes. For example, if themethod of digestion generates blunt ended fragments, the same adaptorsequence can be ligated to both ends. Alternatively some pairs ofenzymes leave identical overhanging sequences. For example, BglIIrecognizes the sequence 5′-AGATCT-3′, cutting after the first A, andBamHI recognizes the sequence 5′-GGATCC-3′, cutting after the first G;both leave an overhang of 5′-GATC-3′. A single adaptor with an overhangof 5′-GATC-3′ may be ligated to both digestion products.

When a single adaptor sequence is ligated to both ends of a fragment theends of a single fragment may be complementary resulting in thepotential formation of hairpin structures. Formation of a base pairinginteraction between the 5′ and 3′ ends of a fragment can inhibitamplification during PCR resulting in lowered overall yield. This effectwill be more pronounced with smaller fragments than with largerfragments because the probability that the ends will hybridize is higherfor smaller fragments than for larger fragments.

Digestion with two or more enzymes can be used to selectively ligateseparate adapters to either end of a restriction fragment. For example,if a fragment is the result of digestion with EcoRI at one end and BamHIat the other end, the overhangs will be 5′-AATT-3′ and 5′-GATC-3′,respectively. An adaptor with an overhang of AATT will be preferentiallyligated to one end while an adaptor with an overhang of GATC will bepreferentially ligated to the second end.

Methods of ligation will be known to those of skill in the art and aredescribed, for example in Sambrook et at. and the New England BioLabscatalog both of which are incorporated herein by reference in theirentireties. Methods include using T4 DNA Ligase which catalyzes theformation of a phosphodiester bond between juxtaposed 5′ phosphate and3′ hydroxyl termini in duplex DNA or RNA with blunt or sticky ends; TaqDNA ligase which catalyzes the formation of a phosphodiester bondbetween juxtaposed 5′ phosphate and 3′ hydroxyl termini of two adjacentoligonucleotides which are hybridized to a complementary target DNA; E.coli DNA ligase which catalyzes the formation of a phosphodiester bondbetween juxtaposed 5′-phosphate and 3′-hydroxyl termini in duplex DNAcontaining cohesive ends; and T4 RNA ligase which catalyzes ligation ofa 5′ phosphoryl-terminated nucleic acid donor to a 3′hydroxyl-terminated nucleic acid acceptor through the formation of a 3′to 5′ phosphodiester bond, substrates include single-stranded RNA andDNA as well as dinucleoside pyrophosphates; or any other substratesdescribed in the art.

A genome is all the genetic material of an organism. In some instances,the term genome may refer to the chromosomal DNA. Genome may bemultichromosomal such that the DNA is cellularly distributed among aplurality of individual chromosomes. For example, in human there are 22pairs of chromosomes plus a gender associated XX or XY pair. DNA derivedfrom the genetic material in the chromosomes of a particular organism isgenomic DNA. The term genome may also refer to genetic materials fromorganisms that do not have chromosomal structure. In addition, the termgenome may refer to mitochondria DNA. A genomic library is a collectionof DNA fragments representing the whole or a portion of a genome.Frequently, a genomic library is a collection of clones made from a setof randomly generated, sometimes overlapping DNA fragments representingthe entire genome or a portion of the genome of an organism.

The term “chromosome” refers to the heredity-bearing gene carrier of acell which is derived from chromatin and which comprises DNA and proteincomponents (especially histones). The conventional internationallyrecognized individual human genome chromosome numbering system isemployed herein. The size of an individual chromosome can vary from onetype to another within a given multi-chromosomal genome and from onegenome to another. In the case of the human genome, the entire DNA massof a given chromosome is usually greater than about 100,000,000 bp. Forexample, the size of the entire human genome is about 3×10⁹ bp. Thelargest chromosome, chromosome no. 1, contains about 2.4×10⁸ bp whilethe smallest chromosome, chromosome no. 22, contains about 5.3×10⁷ bp.

A “chromosomal region” is a portion of a chromosome. The actual physicalsize or extent of any individual chromosomal region can vary greatly.The term “region” is not necessarily definitive of a particular one ormore genes because a region need not take into specific account theparticular coding segments (exons) of an individual gene.

An allele refers to one specific form of a genetic sequence (such as agene) within a cell, an individual or within a population, the specificform differing from other forms of the same gene in the sequence of atleast one, and frequently more than one, variant sites within thesequence of the gene. The sequences at these variant sites that differbetween different alleles are termed “variances”, “polymorphisms”, or“mutations”. At each autosomal specific chromosomal location or “locus”an individual possesses two alleles, one inherited from one parent andone from the other parent, for example one from the mother and one fromthe father. An individual is “heterozygous” at a locus if it has twodifferent alleles at that locus. An individual is “homozygous” at alocus if it has two identical alleles at that locus.

Polymorphism refers to the occurrence of two or more geneticallydetermined alternative sequences or alleles in a population. Apolymorphic marker or site is the locus at which divergence occurs.Preferred markers have at least two alleles, each occurring at afrequency of preferably greater than 1%, and more preferably greaterthan 10% or 20% of a selected population. A polymorphism may compriseone or more base changes, an insertion, a repeat, or a deletion. Apolymorphic locus may be as small as one base pair. Polymorphic markersinclude restriction fragment length polymorphisms, variable number oftandem repeats (VNTR's), hypervariable regions, minisatellites,dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats,simple sequence repeats, and insertion elements such as Alu. The firstidentified allelic form is arbitrarily designated as the reference formand other allelic forms are designated as alternative or variantalleles. The allelic form occurring most frequently in a selectedpopulation is sometimes referred to as the wildtype form. A diallelicpolymorphism has two forms. A triallelic polymorphism has three forms. Apolymorphism between two nucleic acids can occur naturally, or be causedby exposure to or contact with chemicals, enzymes, or other agents, orexposure to agents that cause damage to nucleic acids, for example,ultraviolet radiation, mutagens or carcinogens.

The term genotyping refers to the determination of the geneticinformation an individual carries at one or more positions in thegenome. For example, genotyping may comprise the determination of whichallele or alleles an individual carries for a single SNP or thedetermination of which allele or alleles an individual carries for aplurality of SNPs. For example, a particular nucleotide in a genome maybe an A in some individuals and a C in other individuals. Thoseindividuals who have an A at the position have the A allele and thosewho have a C have the C allele. In a diploid organism the individualwill have two copies of the sequence containing the polymorphic positionso the individual may have an A allele and a C allele or alternativelytwo copies of the A allele or two copies of the C allele. Thoseindividuals who have two copies of the C allele are homozygous for the Callele, those individuals who have two copies of the A allele arehomozygous for the C allele, and those individuals who have one copy ofeach allele are heterozygous. The array may be designed to distinguishbetween each of these three possible outcomes. A polymorphic locationmay have two or more possible alleles and the array may be designed todistinguish between all possible combinations.

Normal cells that are heterozygous at one or more loci may give rise totumor cells that are homozygous at those loci. This loss ofheterozygosity may result from structural deletion of normal genes orloss of the chromosome carrying the normal gene, mitotic recombinationbetween normal and mutant genes, followed by formation of daughter cellshomozygous for deleted or inactivated (mutant) genes; or loss of thechromosome with the normal gene and duplication of the chromosome withthe deleted or inactivated (mutant) gene.

Single nucleotide polymorphisms (SNPs) are positions at which twoalternative bases occur at appreciable frequency (>1%) in a givenpopulation. SNPs are the most common type of human genetic variation. Apolymorphic site is frequently preceded by and followed by highlyconserved sequences (e.g., sequences that vary in less than 1/100 or1/1000 members of the populations).

A SNP may arise due to substitution of one nucleotide for another at thepolymorphic site. A transition is the replacement of one purine byanother purine or one pyrimidine by another pyrimidine. A transversionis the replacement of a purine by a pyrimidine or vice versa. SNPs canalso arise from a deletion of a nucleotide or an insertion of anucleotide relative to a reference allele.

Linkage disequilibrium or allelic association means the preferentialassociation of a particular allele or genetic marker with a specificallele, or genetic marker at a nearby chromosomal location morefrequently than expected by chance for any particular allele frequencyin the population. For example, if locus X has alleles a and b, whichoccur at equal frequency, and linked locus Y has alleles c and d, whichoccur at equal frequency, one would expect the combination ac to occurat a frequency of 0.25. If ac occurs more frequently, then alleles a andc are in linkage disequilibrium. Linkage disequilibrium may result, forexample, because the regions are physically close, from naturalselection of certain combination of alleles or because an allele hasbeen introduced into a population too recently to have reachedequilibrium with linked alleles. A marker in linkage disequilibrium canbe particularly useful in detecting susceptibility to disease (or otherphenotype) notwithstanding that the marker does not cause the disease.For example, a marker (X) that is not itself a causative element of adisease, but which is in linkage disequilibrium with a gene (includingregulatory sequences) (Y) that is a causative element of a phenotype,can be detected to indicate susceptibility to the disease incircumstances in which the gene Y may not have been identified or maynot be readily detectable.

The term “target sequence”, “target nucleic acid” or “target” refers toa nucleic acid of interest. The target sequence may or may not be ofbiological significance. Typically, though not always, it is thesignificance of the target sequence which is being studied in aparticular experiment. As non-limiting examples, target sequences mayinclude regions of genomic DNA which are believed to contain one or morepolymorphic sites, DNA encoding or believed to encode genes or portionsof genes of known or unknown function, DNA encoding or believed toencode proteins or portions of proteins of known or unknown function,DNA encoding or believed to encode regulatory regions such as promotersequences, splicing signals, polyadenylation signals, etc. In manyembodiments a collection of target sequences is identified and assayed.

A sequence may be selected to be a target sequence if it has a region ofinterest and shares a characteristic with at least one other targetsequence that will allow the two or more target sequences to be enrichedin a subset of fragments. The region of interest may be, for example, asingle nucleotide polymorphism and the shared characteristic may be, forexample, that the target sequence is found on a fragment in a selectedsize range when a genomic sample is fragmented by digestion with aparticular enzyme or enzyme combination. Collections of target sequencesthat each have a region of interest and share a common characteristicare particularly useful. For example, a collection of target sequencesthat each contain a location that is known to be polymorphic in apopulation and are each found on a fragment that is between 400 and 800base pairs when human genomic DNA is digested with XbaI may beinterrogated in a single assay. A collection may comprise from 2, 100,1,000, 5,000, 10,000, or 50,000 to 1,000, 5,000, 10,000, 20,000, 50,000,100,000, 1,000,000 or 3,500,000 different target sequences.

The term subset of fragments or representative subset refers to afraction of a genome. The subset may be less than or about 0.01, 0.1, 1,3, 5, 10, 25, 50 or 75% of the genome. The partitioning of fragmentsinto subsets may be done according to a variety of physicalcharacteristics of individual fragments. For example, fragments may bedivided into subsets according to size, according to the particularcombination of restriction sites at the ends of the fragment, or basedon the presence or absence of one or more particular sequences.

Target sequences may be interrogated by hybridization to an array. Thearray may be specially designed to interrogate one or more selectedtarget sequence. The array may contain a collection of probes that aredesigned to hybridize to a region of the target sequence or itscomplement. Different probe sequences are located at spatiallyaddressable locations on the array. For genotyping a single polymorphicsite probes that match the sequence of each allele may be included. Atleast one perfect match probe, which is exactly complementary to thepolymorphic base and to a region surrounding the polymorphic base, maybe included for each allele. Multiple perfect match probes may beincluded as well as mismatch probes.

An “array” comprises a support, preferably solid, with nucleic acidprobes attached to the support. Preferred arrays typically comprise aplurality of different nucleic acid probes that are coupled to a surfaceof a substrate in different, known locations. These arrays, alsodescribed as “microarrays” or colloquially “chips” have been generallydescribed in the art, for example, U.S. Pat. Nos. 5,143,854, 5,445,934,5,744,305, 5,677,195, 5,800,992, 6,040,193, 5,424,186 and Fodor et al.,Science, 251:767-777 (1991), each of which is incorporated by referencein its entirety for all purposes.

Arrays may generally be produced using a variety of techniques, such asmechanical synthesis methods or light directed synthesis methods thatincorporate a combination of photolithographic methods and solid phasesynthesis methods.

Techniques for the synthesis of these arrays using mechanical synthesismethods are described in, e.g., U.S. Pat. Nos. 5,384,261, and 6,040,193,which are incorporated herein by reference in their entirety for allpurposes. Although a planar array surface is preferred, the array may befabricated on a surface of virtually any shape or even a multiplicity ofsurfaces. Arrays may be nucleic acids on beads, gels, polymericsurfaces, fibers such as fiber optics, glass or any other appropriatesubstrate. (See U.S. Pat. Nos. 5,770,358, 5,789,162, 5,708,153,6,040,193 and 5,800,992, which are hereby incorporated by reference intheir entirety for all purposes.) Arrays may be packaged in such amanner as to allow for diagnostic use or can be an all-inclusive device;e.g., U.S. Pat. Nos. 5,856,174 and 5,922,591 incorporated in theirentirety by reference for all purposes.

Preferred arrays are commercially available from Affymetrix (SantaClara, Calif.) under the brand name GeneChip® and are directed to avariety of purposes, including genotyping and gene expression monitoringfor a variety of eukaryotic and prokaryotic species.

Hybridization probes are oligonucleotides capable of binding in abase-specific manner to a complementary strand of nucleic acid. Suchprobes include peptide nucleic acids, as described in Nielsen et al.,Science 254, 1497-1500 (1991), and other nucleic acid analogs andnucleic acid mimetics. See U.S. patent application Ser. No. 08/630,427.

Hybridizations are usually performed under stringent conditions, forexample, at a salt concentration of no more than 1 M and a temperatureof at least 25° C. For example, conditions of 5×SSPE (750 mM NaCl, 50 mMNaPhosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30° C. aresuitable for allele-specific probe hybridizations. For stringentconditions, see, for example, Sambrook et al. which is herebyincorporated by reference in its entirety for all purposes above.

An individual is not limited to a human being, but may also includeother organisms including but not limited to mammals, plants, fungi,bacteria or cells derived from any of the above.

(C.) Preferential Amplification of a Subset of Fragments ContainingTarget Sequences

The present invention provides for novel methods of analysis of anucleic acid sample, such as genomic DNA. The methods include:identification and selection of a collection of target sequences;amplification of a selected subset of fragments that comprises acollection of target sequences; and, analysis of a collection of targetsequences. In many embodiments a subset of fragments may be amplified byPCR wherein the subset of fragments that is amplified efficiently isdependent on the size of the fragments. In one embodiment fragmentationconditions and target sequences are selected so that the targetsequences are present in the subset of fragments that are efficientlyamplified by PCR. Those fragments that are efficiently amplified areenriched in the amplified sample and are present in amounts sufficientfor hybridization analysis and detection using the methods disclosed.Many fragments will not be amplified efficiently enough for detectionusing the methods disclosed and these fragments are not enriched in theamplified sample.

In many embodiments the methods include the steps of: identifying acollection of target sequences that carry sequences of interest onfragments of a selected size range; fragmenting a nucleic acid sample bydigestion with one or more restriction enzymes so that the targetsequences are present on fragments that are within the selected sizerange; ligating one or more adaptors to the fragments; and amplifyingthe fragments so that a subset of the fragments, including fragments ofthe selected size range, are enriched in the amplified product. In someembodiments the amplified sample is exposed to an array which may bespecifically designed and manufactured to interrogate one or more targetsequences in a collection of target sequences.

In some embodiments the selected size range is selected to be within thesize range of fragments that can be efficiently amplified under a givenset of amplification conditions. In many embodiments amplification is byPCR and the PCR conditions are standard PCR amplification conditions(see, for example, PCR primer A laboratory Manual, Cold Spring HarborLab Press, (1995) eds. C. Dieffenbach and G. Dveksler), under theseconditions fragments that are of a predicted size range, generally lessthan 2 kb, will be amplified most efficiently.

FIG. 1 illustrates an example of how possible target sequences may bedefined. The starting set is all of the fragments of the genomefollowing fragmentation. Within this set there is a subset of fragmentsthat are about 2 kb and less and would be efficiently amplified by PCRunder standard conditions. Also within the starting set is a subset offragments that contain sequences of interest, for example, fragmentsthat contain SNPs. There is an intersection between these two subsetsthat represents fragments that will be efficiently amplified understandard PCR conditions and contain sequences of interest. In oneembodiment these fragments are possible target sequences. In someembodiments a smaller subset is selected from within the subset offragments that are about 2 kb and less. This subset may be, for example,fragments from about 1, 100, 200, or 400 bp to 600, 800, 1,200, 1,500 or2,000 bp. The intersection of this subset with the subset of fragmentsthat comprise a sequence of interest contains fragments that arepotential target sequences. The set of potential target sequences willvary depending on the fragmentation method used and the size range thatis selected. The collection of target sequences may comprise allpotential target sequences or a further subset of the possible targetsequences. Potential target sequences may be selected for the collectionof target sequences or removed from the collection of target sequencesbased on secondary considerations such as performance in hybridizationexperiments, location in the genome, proximity to another targetsequence in the collection, association with phenotype or disease or anyother criteria that is known in the art. Additional selection criteriathat may be used to select target sequences for a collection of targetsequences also include, for example, clustering characteristics, whetheror not a SNP is consistently present in a population, Mendelianinheritance characteristics, Hardy-Weinberg probability, and chromosomalmap distribution. In one embodiment fragments that contain repetitivesequences, telomeric regions, centromeric regions and heterochromatindomains may be excluded. In one embodiment the target sequences compriseSNPs and the SNPs are selected to provide an optimal representation ofthe genome. For example SNPs may be selected so that the distancebetween SNPs in the target collection is on average between 10, 50, 100,200 or 300 and 50, 100, 200, 400, 600 or 800 kb. Inter-SNP distances mayvary from chromosome to chromosome. In one embodiment more than 80% ofthe SNPs in the collection of target sequence are less than about 200 kbfrom another SNP in the collection of target sequences. In anotherembodiment more than 80% of the SNPs are less than about 10, 50, 100,150, 300 or 500 kb from another SNP in the collection of targetsequences. In one embodiment SNPs that give errors across multiplefamilies are not selected for the collection of SNPs or are removed fromthe analysis. In another embodiment SNPs that give ambiguous results inmultiple experiments are not selected for the collection of SNPs or areremoved from the analysis.

In many embodiments the methods employ the use of a computer system toassist in the identification of potential target sequences or in theselection of target sequences for a collection. For many organisms,including yeast, mouse, human and a number of microbial species, acomplete or complete draft of the genomic sequence is known and publiclyavailable. Knowledge of the sequences present in a nucleic acid sample,such as a genome, allow prediction of the sizes and sequence content offragments that will result when the genome is fragmented under selectedconditions. The pool of predicted fragments may be analyzed to identifywhich fragments are within a selected size range, which fragments carrya sequence of interest and which fragments have both characteristics. Insome embodiments an array may then be designed to interrogate at leastsome of those potential target sequences. A nucleic acid sample may thenbe digested with the selected enzyme or enzyme and amplified under theselected amplification conditions, resulting in the amplification of thecollection of target sequences. The amplified target sequences may thenbe analyzed by hybridization to the array. In some embodiments theamplified sequences may be further analyzed using any known methodincluding sequencing, HPLC, hybridization analysis, cloning, labeling,etc.

In many embodiments in silico digestion techniques are used to identifyone or more SNPs that will be present on fragments of a selected sizewhen a genome is digested with a particular enzyme or enzymecombination. In FIG. 2, a computer is used to locate a SNP from a publicdatabase, for example the database provided by The SNP Consortium (TSC),or within the sequence of the human genome, for example in the publiclyavailable database such as Genbank. A computer may then be used topredict the, for example, BglII restriction sites upstream anddownstream of a given SNP. For example, in FIG. 2 TSC SNP ID 10034 has aBglII site at upstream position 49050 and a downstream BglII site atposition 52100. Given these restriction sites it is predicted that SNP10034 will be contained on a 3050 base pair fragment when human genomicDNA is digested with BglII.

The SNPs and corresponding fragment sizes may be further separated intosubsets according to fragment size. In some embodiment this step isperformed by a computer or computer system. In this way a computer couldbe used to identify all of the SNPs that are predicted to be found onfragments that are between, for example, 200, 400, 600 or 800 and 800,1000, 1500 or 2000 base pairs in length when a sample DNA is digestedwith a selected enzyme or enzyme combination.

In another embodiment the SNPs present on fragments of a selected sizerange following fragmentation are selected as target sequences and anarray is designed to interrogate at least some of the SNPs. For example,an array may be designed to genotype some of the SNPs that are presenton fragments of 400 to 800 base pairs when human genomic DNA is digestedwith XbaI. If, for example, there are 15,000 SNPs that meet thesecriteria a subset of these SNP, for example, 10,000 may be selected forthe array.

In FIG. 3, in silico digestion was used to predict restriction fragmentlengths for the more than 800,000 SNPs in the TSC database and toidentify those SNP containing fragments between 400 and 800 base pairs.For example, when human genomic DNA is digested with EcoRI, 32,908 SNPsfrom the TSC database are predicted to be found on fragments between 400and 800 base pairs. More than 120,000 of the TSC SNPs are found onfragments between 400 and 800 base pairs when genomic DNA is digestedwith EcoRI, XbaI, PstI and BglII.

In one embodiment in silico prediction of the size of SNP containingfragments is combined with selection of a collection of target sequencesto design genotyping assays and arrays for genotyping, see FIG. 4. Inone embodiment target sequences are selected from fragments that arethose in the size range of 400 to 800 base pairs, but other size rangescould also be used, for example, 100, 200, 500, 700, or 1,500 to 500,700, 1,000, or 2,000 base pairs may also be useful size ranges.

As shown in FIG. 4, in this embodiment an array is designed tointerrogate the SNPs that are predicted to be found in a size fractionresulting from digestion of the first nucleic acid sample with one ormore particular restriction enzymes. For example, a computer may be usedto search the sequence of a genome to identify all recognition sites forthe restriction enzyme, EcoRI. The computer can then be used to predictthe size of all restriction fragments resulting from an EcoRI digestionand to identify those fragments that contain a known or suspected SNP orpolymorphism. The computer may then be used to identify the group ofSNPs that are predicted to be found on fragments of, for example,400-800 base pairs, when genomic DNA is digested with EcoRI. An arraymay then be designed to interrogate that subset of SNPs that are foundon EcoRI fragments of 400-800 base pairs.

Arrays will preferably be designed to interrogate from 100, 500, 1000,5000, 8000, 10,000, or 50,000 to 5,000, 10,000, 15,000, 30,000,100,000,500,000 or 1,500,000 different SNPs. For example, an array may bedesigned to interrogate a collection of target sequences comprising acollection of SNPs predicted to be present on 400-800 base pair EcoRIfragments, a collection of SNPs predicted to be present on 400-800 basepair BglII fragments, a collection of SNPs predicted to be present on400-800 base pair XbaI fragments, and a collection of SNPs predicted tobe present on 400-800 base pair HindIII fragments. One or more amplifiedsubsets of fragments may be pooled prior to hybridization to increasethe complexity of the sample.

In some embodiments a single size selected amplification product issuitable for hybridization to many different arrays. For example, asingle method of fragmentation and amplification that is suitable forhybridization to an array designed to interrogate SNPs contained on400-800 base pair EcoRI would also be suitable for hybridization to anarray designed to interrogate SNPs contained on 400-800 base pair BamHIfragments. This would introduce consistency and reproducibility tosample preparation methods.

In some embodiments SNPs present in a collection of target sequences arefurther characterized and an array is designed to interrogate a subsetof these SNPs. SNPs may be selected for inclusion on an array based on avariety of characteristics, such as, for example, allelic frequency in apopulation, distribution in a genome, hybridization performance,genotyping performance, number of probes necessary for accurategenotyping, available linkage information, available mappinginformation, phenotypic characteristics or any other information about aSNP that makes it a better or worse candidate for analysis.

In many embodiments a selected collection of target sequences may beamplified reproducibly from different samples or from the same sample indifferent reactions. In one embodiment a plurality of samples areamplified in different reactions under similar conditions and eachamplification reaction results in amplification of a similar collectionof target sequences. Genomic samples from different individuals may befragmented and amplified using a selected set of conditions and similartarget sequences will be amplified from both samples. For example, ifgenomic DNA is isolated from 2 or more individuals, each sample isfragmented under similar conditions, amplified under similar conditionsand hybridized to arrays designed to interrogate the same collection oftarget sequences, more than 50%, more than 75% or more than 90% of thesame target sequences are detected in the samples.

A given target sequence may be present in different allelic forms in acell, a sample, an individual and in a population. In some embodimentsthe methods identify which alleles are present in a sample. In someembodiments the methods determine heterozygosity or homozygosity at oneor more loci. In some embodiments, where SNPs are being interrogated forgenotype, a genotype is determined for more than 75%, 85% or 90% of theSNPs interrogated by the array. In some embodiments the hybridizationpattern on the array is analyzed to determine a genotype. In someembodiments analysis of the hybridization is done with a computer systemand the computer system provides a determination of which alleles arepresent.

In one embodiment target sequences are selected from the subset offragments that are less than 1,000 base pairs. An in silico digestion ofthe human genome may be used to identify fragments that are less than1,000 base pairs when the genome is digested with the restrictionenzyme, XbaI. The predicted XbaI fragments that are under 1,000 basepairs may be analyzed to identify SNPs that are present on thefragments. An array may be designed to interrogate the SNPs present onthe fragments and the probes may be designed to determine which allelesof the SNP are present. A genomic sample may be isolated from anindividual, digested with XbaI, adaptors are ligated to the fragmentsand the fragments are amplified. The amplified sample may be hybridizedto the specially designed array and the hybridization pattern may beanalyzed to determine which alleles of the SNPs are present in thesample from this individual.

In some embodiments the size range of fragments remains approximatelyconstant, and the target sequences present in the size range vary withthe method of fragmentation used. For example, if the target sequencesare SNP containing fragments that are 400-800 base pairs, the fragmentsthat meet these criteria when the human genome is digested with XbaIwill be different than when the genome is digested with EcoRI, althoughthere may be some overlap. By using a different fragmentation method butkeeping the amplification conditions constant different collections oftarget sequences may be analyzed. In some embodiments an array may bedesigned to interrogate target sequences resulting from just onefragmentation condition and in other embodiments the array may bedesigned to interrogate fragments resulting from more than onefragmentation condition. For example, an array may be designed tointerrogate the SNPs present on fragments that are less than 1,000 basepairs when a genome is digested with XbaI and the SNPs present onfragments that are less than 1,000 base pairs when a genome is digestedwith EcoRI.

In many embodiments an enzyme is selected so that digestion of thesample with the selected enzyme followed by amplification results in asample of a complexity that may be specifically hybridized to an arrayunder selected conditions. For example, digestion of the human genomewith XbaI, EcoRI or BglII and amplification with PCR reduces complexityof the sample to approximately 2%. In another embodiment the sample isdigested with an enzyme that cuts the genome at frequencies similar toXbaI, EcoRI or BglII, for example, SacI, BsrGI or BclI. Differentcomplexity levels may be used. Useful complexities range from 0.1, 2, 5,10 or 25% to 1, 2, 10, 25 or 50% of the complexity of the startingsample. In some embodiments the complexity of the sample is matched tothe content on an array.

In many embodiments the target sequences are a subset that isrepresentative of a larger set. For example, the target sequences may be1,000, 5,000, 10,000 or 100,000 to 10,000, 20,000, 100,000, 1,500,000 or3,000,000 SNPs that may be representative of a larger population of SNPspresent in a population of individuals. The target sequences may bedispersed throughout a genome, including for example, sequences fromeach chromosome, or each arm of each chromosome. Target sequences may berepresentative of haplotypes or particular phenotypes or collections ofphenotypes. For a description of haplotypes see, for example, Gabriel etal., Science, 296:2225-9 (2002), Daly et al. Nat Genet., 29:229-32(2001) and Rioux et al., Nat Genet., 29:223-8 (2001), each of which isincorporated herein by reference in its entirety.

The methods may be combined with other methods of genome analysis andcomplexity reduction. Other methods of complexity reduction include, forexample, AFLP, see U.S. Pat. No. 6,045,994, which is incorporated hereinby reference, and arbitrarily primed-PCR (AP-PCR) see McClelland andWelsh, in PCR Primer: A laboratory Manual, (1995) eds. C. Dieffenbachand G. Dveksler, Cold Spring Harbor Lab Press, for example, at p 203,which is incorporated herein by reference in its entirety. Additionalmethods of sample preparation and techniques for reducing the complexityof a nucleic sample are described in Dong et al., Genome Research 11,1418 (2001), in U.S. Pat. Nos. 6,361,947, 6,391,592 and U.S. patentapplication Ser. Nos. 09/512,300, 09/916,135, 09/920,491, 09/910,292,and 10/013,598, which are incorporated herein by reference in theirentireties.

One method that has been used to isolate a subset of a genome is toseparate fragments according to size by electrophoresis in a gel matrix.The region of the gel containing fragments in the desired size range isthen excised and the fragments are purified away from the gel matrix.The SNP consortium (TSC) adopted this approach in their efforts todiscover single nucleotide polymorphisms (SNPs) in the human genome.See, Altshuler et al., Science 407: 513-516 (2000) and The InternationalSNP Map Working Group, Nature 409: 928-933 (2001) both of which areherein incorporated by reference in their entireties for all purposes.

PCR amplification of a subset of fragments is an alternative,non-gel-based method to reduce the complexity of a sample. PCRamplification in general is a method of reducing the complexity of asample by preferentially amplifying one or more sequences from a complexsample. This effect is most obvious when locus specific primers are usedto amplify a single sequence from a complex sample, but it is alsoobserved when a collection of sequences is targeted for amplification.

There are many known methods of amplifying nucleic acid sequencesincluding e.g., PCR. See, e.g., PCR Technology: Principles andApplications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY,N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (eds.Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al.,Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods andApplications 1, 17 (1991); PCR (eds. McPherson et al., IRL Press,Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, 4,965,188and 5,333,675 each of which is incorporated herein by reference in theirentireties for all purposes.

PCR is an extremely powerful technique for amplifying specificpolynucleotide sequences, including genomic DNA, single-stranded cDNA,and mRNA among others. Various methods of conducting PCR amplificationand primer design and construction for PCR amplification will be knownto those of skill in the art. Generally, in PCR a double stranded DNA tobe amplified is denatured by heating the sample. New DNA synthesis isthen primed by hybridizing primers to the target sequence in thepresence of DNA polymerase and excess dNTPs. In subsequent cycles, theprimers hybridize to the newly synthesized DNA to produce discreetproducts with the primer sequences at either end. The productsaccumulate exponentially with each successive round of amplification.

The DNA polymerase used in PCR is often a thermostable polymerase. Thisallows the enzyme to continue functioning after repeated cycles ofheating necessary to denature the double stranded DNA. Polymerases thatare useful for PCR include, for example, Taq DNA polymerase, Tth DNApolymerase, Tfl DNA polymerase, Tma DNA polymerase, Tli DNA polymerase,and Pfu DNA polymerase. There are many commercially available modifiedforms of these enzymes including: AmpliTaq®, AmpliTaq® Stoffel Fragmentand AmpliTaq Gold® available from Applied Biosystems (Foster City,Calif.). Many are available with or without a 3- to 5′ proofreadingexonuclease activity. See, for example, Vent® and Vent® (exo-) availablefrom New England Biolab (Beverly, Mass.).

Other suitable amplification methods include the ligase chain reaction(LCR) (e.g., Wu and Wallace, Genomics 4, 560 (1989) and Landegren etal., Science 241, 1077 (1988)), transcription amplification (Kwoh etal., Proc. Natl. Acad. Sci. USA 86, 1173 (1989)), self-sustainedsequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87,1874 (1990)) and nucleic acid based sequence amplification (NABSA).(See, U.S. Pat. Nos. 5,409,818, 5,554,517, and 6,063,603 each of whichis incorporated herein by reference in their entireties).

When genomic DNA is digested with one or more restriction enzymes thesizes of the fragments are randomly distributed over a broad range.Following adaptor ligation, all of the fragments that have adaptorsligated to both ends will compete equally for primer binding andextension regardless of size. However, standard PCR typically results inmore efficient amplification of fragments that are smaller than 2.0 kb.(See Saiki et al. Science 239, 487-491 (1988) which is herebyincorporated by reference it its entirety). The natural tendency of PCRis to amplify shorter fragments more efficiently than longer fragments.This inherent length dependence of PCR results in efficientamplification of only a subset of the starting fragments. Thosefragments that are smaller than 2 kb will be more efficiently amplifiedthan larger fragments when a standard range of conditions are used. Thiseffect may be related to the processivity of the enzyme, which limitsthe yield of polymerization products over a given unit of time. Thepolymerase may also fail to complete extension of a given template if itfalls off the template prior to completion. What is observed is thatlonger templates are less efficiently amplified under a standard rangeof PCR conditions than shorter fragments. Because of the geometricnature of PCR amplification, subtle differences in yields that occur inthe initial cycles will result in significant differences in yields inlater cycles. (See, PCR Primer: A Laboratory Manual, CSHL Press, Eds.Carl Dieffenbach and Gabriela Dveskler, (1995), (Dieffenbach et al.)which is herein incorporated by reference in its entirety for allpurposes.) Variations in the reaction conditions such as, for example,primer concentration, extension time, salt concentration, buffer,temperature, and number of cycles may alter the size distribution offragments to some extent. Inclusion of chain terminating nucleotides ornucleotide analogs may also alter the subset of fragments that areamplified. (See, Current Protocols in Molecular Biology, eds. Ausubel etal. (2000), which is herein incorporated by reference in its entiretyfor all purposes.) The presence or absence of exonuclease activity mayalso be used to modify the subset of fragments amplified. (See, forexample, PCR Strategies, eds. Innis et al, Academic Press (1995), (Inniset al.), which is herein incorporated by reference for all purposes).

Ligation of a single adapter sequence to both ends of the fragments mayalso impact the efficiency of amplification of smaller fragments due tothe formation of pan-handle structures between the resulting terminalrepeats, see, for example, Qureshi et al. GATA 11(4): 95-101, (1994),Caetano-Anolles et al. Mol. Gen. Genet. 235: 157-165 (1992) and Jonesand Winistorfer, PCR Methods Appl. 2:197-203 (1993). Smaller fragmentsare more likely to form the pan-handle structure and the loop may bemore stable than longer loops.

In some embodiments sequences that are on smaller fragments, forexample, fragments less than 400 bp or less than 200 bp are not selectedas target sequences. In addition to the bias against amplification ofthese fragments when a single adapter is used there are also fewer smallfragments following fragmentation with a restriction enzyme or enzymes.For many enzymes fragments that are, for example, smaller than 200 basepairs are relatively rare in the sample being amplified because of theinfrequency of the recognition site. Since the small fragments are rareand account for relatively little sequence information there is also adecreased probability that sequences of interest will be present onsmall fragments.

In some embodiments the potential for formation of a stable duplexbetween the ends of the fragment strands is reduced. In one embodimentthe adapter contains internal mismatches. In another embodiment the twostrands of the adapter have a region of complementarity and a region ofnon-complementarity (FIG. 5). The region of complementarity (A and A′)is near the end that will ligate to the fragments. The fragments can beamplified using primers to the non-complementary regions (B and C).Amplified products will have sequences B and C at the ends which willdestabilize basepairing between A and A′. In another embodiment thesample may be amplified with a single primer for at least some of thecycles of amplification.

In some embodiments two or more different adaptors are ligated to theends of the fragments. Ligation of different adaptor sequences to thefragments may result in some fragments that have the same adaptorligated to both ends and some fragments that have two different adaptorsligated to each end. Small fragments that have different adaptorsligated to each end are more efficient templates for amplification thansmall fragments that have the same adaptor ligated to both ends becausethe potential for base pairing between the ends of the fragments iseliminated or reduced.

In one embodiment, the fragmented sample is fractionated prior toamplification by, for example, applying the sample to a gel exclusioncolumn. Adaptors may be ligated to the fragments before or afterfractionation. For example, to exclude the shortest fragments from theamplification the fragments can be passed over a column that selectivelyretains smaller fragments, for example fragments under 400 base pairs.The larger fragments may be recovered in the void volume. Because theshortest fragments in the PCR would be approximately 400 base pairs, theresulting PCR products will primarily be in a size range larger than 400base pairs.

The materials for use in the present invention are ideally suited forthe preparation of a kit suitable for obtaining an amplified collectionof target sequences. Such a kit may comprise various reagents utilizedin the methods, preferably in concentrated form. The reagents of thiskit may comprise, but are not limited to, buffer, appropriate nucleotidetriphosphates, appropriate dideoxynucleotide triphosphates, reversetranscriptases, nucleases, restriction enzymes, adaptors, ligases, DNApolymerases, primers, instructions for the use of the kit and arrays.

In order to interrogate a whole genome it is often useful to amplify andanalyze one or more representative subsets of the genome. There may bemore than 3,000,000 SNPs in the human genome, but tremendous amounts ofinformation may be obtained by analysis of a subset of SNPs that isrepresentative of the whole genome. Subsets can be defined by manycharacteristics of the fragments. In a preferred embodiment of thecurrent invention, the subsets are defined by the proximity to anupstream and downstream restriction site and by the size of thefragments resulting from restriction enzyme digestion. Useful sizeranges may be from 100, 200, 400, 700 or 1000 to 500, 800, 1500, 2000,4000 or 10,000. However, larger size ranges such as 4000, 10,000 or20,000 to 10,000, 20,000 or 500,000 base pairs may also be useful.

The disclosed methods may be applied to many different organismsincluding plants, bacteria and animals, including, human, mouse, rat,and dog. Organisms whose genomes have been sequenced are particularlyuseful. The genomic DNA sample may be isolated according to methodsknown in the art. It may be obtained from any biological orenvironmental source, including plant, animal (including human),bacteria, fungi or algae. Any suitable biological sample may be used forassay of genomic DNA. Convenient suitable samples include whole blood,tissue, semen, saliva, tears, urine, fecal material, sweat, buccal, skinand hair.

Methods of Use

The methods of the presently claimed invention can be used for a widevariety of applications including, for example, linkage and associationstudies, identification of candidate gene regions, genotyping clinicalpopulations, correlation of genotype information to phenotypeinformation, loss of heterozygosity analysis, and identification of thesource of an organism or sample, or the population from which anorganism or sample originates. Any analysis of genomic DNA may bebenefited by a reproducible method of complexity management.Furthermore, the methods and enriched fragments of the presently claimedinvention are particularly well suited for study and characterization ofextremely large regions of genomic DNA.

In a preferred embodiment, the methods of the presently claimedinvention are used for SNP discovery and to genotype individuals. Forexample, any of the procedures described above, alone or in combination,could be used to isolate the SNPs present in one or more specificregions of genomic DNA. Selection probes could be designed andmanufactured to be used in combination with the methods of the inventionto amplify only those fragments containing regions of interest, forexample a region known to contain a SNP. Arrays could be designed andmanufactured on a large scale basis to interrogate only those fragmentscontaining the regions of interest. Thereafter, a sample from one ormore individuals would be obtained and prepared using the sametechniques which were used to prepare the selection probes or to designthe array. Each sample can then be hybridized to an array and thehybridization pattern can be analyzed to determine the genotype of eachindividual or a population of individuals. Methods of use forpolymorphisms and SNP discovery can be found in, for example, U.S. Pat.No. 6,361,947 which is herein incorporated by reference in its entiretyfor all purposes.

Correlation of Polymorphisms with Phenotypic Traits

Most human sequence variation is attributable to or correlated withSNPs, with the rest attributable to insertions or deletions of one ormore bases, repeat length polymorphisms and rearrangements. On average,SNPs occur every 1,000-2,000 bases when two human chromosomes arecompared. (See, The International SNP Map Working Group, Science 409:928-933 (2001) incorporated herein by reference in its entirety for allpurposes.) Human diversity is limited not only by the number of SNPsoccurring in the genome but further by the observation that specificcombinations of alleles are found at closely linked sites.

Correlation of individual polymorphisms or groups of polymorphisms withphenotypic characteristics is a valuable tool in the effort to identifyDNA variation that contributes to population variation in phenotypictraits. Phenotypic traits include physical characteristics, risk fordisease, and response to the environment. Polymorphisms that correlatewith disease are particularly interesting because they representmechanisms to accurately diagnose disease and targets for drugtreatment. Hundreds of human diseases have already been correlated withindividual polymorphisms but there are many diseases that are known tohave an, as yet unidentified, genetic component and many diseases forwhich a component is or may be genetic.

Many diseases may correlate with multiple genetic changes makingidentification of the polymorphisms associated with a given disease moredifficult. One approach to overcome this difficulty is to systematicallyexplore the limited set of common gene variants for association withdisease.

To identify correlation between one or more alleles and one or morephenotypic traits, individuals are tested for the presence or absence ofpolymorphic markers or marker sets and for the phenotypic trait ortraits of interest. The presence or absence of a set of polymorphisms iscompared for individuals who exhibit a particular trait and individualswho exhibit lack of the particular trait to determine if the presence orabsence of a particular allele is associated with the trait of interest.For example, it might be found that the presence of allele A1 atpolymorphism A correlates with heart disease. As an example of acorrelation between a phenotypic trait and more than one polymorphism,it might be found that allele A1 at polymorphism A and allele B1 atpolymorphism B correlate with a phenotypic trait of interest.

Diagnosis of Disease and Predisposition to Disease

Markers or groups of markers that correlate with the symptoms oroccurrence of disease can be used to diagnose disease or predispositionto disease without regard to phenotypic manifestation. To diagnosedisease or predisposition to disease, individuals are tested for thepresence or absence of polymorphic markers or marker sets that correlatewith one or more diseases. If, for example, the presence of allele A1 atpolymorphism A correlates with coronary artery disease then individualswith allele A1 at polymorphism A may be at an increased risk for thecondition.

Individuals can be tested before symptoms of the disease develop.Infants, for example, can be tested for genetic diseases such asphenylketonuria at birth. Individuals of any age could be tested todetermine risk profiles for the occurrence of future disease. Oftenearly diagnosis can lead to more effective treatment and prevention ofdisease through dietary, behavior or pharmaceutical interventions.Individuals can also be tested to determine carrier status for geneticdisorders. Potential parents can use this information to make familyplanning decisions.

Individuals who develop symptoms of disease that are consistent withmore than one diagnosis can be tested to make a more accurate diagnosis.If, for example, symptom S is consistent with diseases X, Y or Z butallele A1 at polymorphism A correlates with disease X but not withdiseases Y or Z an individual with symptom S is tested for the presenceor absence of allele A1 at polymorphism A. Presence of allele A1 atpolymorphism A is consistent with a diagnosis of disease X. Geneticexpression information discovered through the use of arrays has beenused to determine the specific type of cancer a particular patient has.(See, Golub et al. Science 286: 531-537 (2001) hereby incorporated byreference in its entirety for all purposes.)

Pharmacogenomics

Pharmacogenomics refers to the study of how genes affect response todrugs. There is great heterogeneity in the way individuals respond tomedications, in terms of both host toxicity and treatment efficacy.There are many causes of this variability, including: severity of thedisease being treated; drug interactions; and the individuals age andnutritional status. Despite the importance of these clinical variables,inherited differences in the form of genetic polymorphisms can have aneven greater influence on the efficacy and toxicity of medications.Genetic polymorphisms in drug-metabolizing enzymes, transporters,receptors, and other drug targets have been linked to interindividualdifferences in the efficacy and toxicity of many medications. (See,Evans and Relling, Science 286: 487-491 (2001) which is hereinincorporated by reference for all purposes).

An individual patient has an inherited ability to metabolize, eliminateand respond to specific drugs. Correlation of polymorphisms withpharmacogenomic traits identifies those polymorphisms that impact drugtoxicity and treatment efficacy. This information can be used by doctorsto determine what course of medicine is best for a particular patientand by pharmaceutical companies to develop new drugs that target aparticular disease or particular individuals within the population,while decreasing the likelihood of adverse affects. Drugs can betargeted to groups of individuals who carry a specific allele or groupof alleles. For example, individuals who carry allele A1 at polymorphismA may respond best to medication X while individuals who carry allele A2respond best to medication Y. A trait may be the result of a singlepolymorphism but will often be determined by the interplay of severalgenes.

In addition some drugs that are highly effective for a large percentageof the population, prove dangerous or even lethal for a very smallpercentage of the population. These drugs typically are not available toanyone. Pharmacogenomics can be used to correlate a specific genotypewith an adverse drug response. If pharmaceutical companies andphysicians can accurately identify those patients who would sufferadverse responses to a particular drug, the drug can be made availableon a limited basis to those who would benefit from the drug.

Similarly, some medications may be highly effective for only a verysmall percentage of the population while proving only slightly effectiveor even ineffective to a large percentage of patients. Pharmacogenomicsallows pharmaceutical companies to predict which patients would be theideal candidate for a particular drug, thereby dramatically reducingfailure rates and providing greater incentive to companies to continueto conduct research into those drugs.

Determination of Relatedness

There are many circumstances where relatedness between individuals isthe subject of genotype analysis and the present invention can beapplied to these procedures. Paternity testing is commonly used toestablish a biological relationship between a child and the putativefather of that child. Genetic material from the child can be analyzedfor occurrence of polymorphisms and compared to a similar analysis ofthe putative father's genetic material. Determination of relatedness isnot limited to the relationship between father and child but can also bedone to determine the relatedness between mother and child, (see e.g.Staub et al., U.S. Pat. No. 6,187,540) or more broadly, to determine howrelated one individual is to another, for example, between races orspecies or between individuals from geographically separatedpopulations, (see for example H. Kaessmann, et al. Nature Genet. 22, 78(1999)).

Forensics

The capacity to identify a distinguishing or unique set of forensicmarkers in an individual is useful for forensic analysis. For example,one can determine whether a blood sample from a suspect matches a bloodor other tissue sample from a crime scene by determining whether the setof polymorphic forms occupying selected polymorphic sites is the same inthe suspect and the sample. If the set of polymorphic markers does notmatch between a suspect and a sample, it can be concluded (barringexperimental error) that the suspect was not the source of the sample.If the set of markers does match, one can conclude that the DNA from thesuspect is consistent with that found at the crime scene. If frequenciesof the polymorphic forms at the loci tested have been determined (e.g.,by analysis of a suitable population of individuals), one can perform astatistical analysis to determine the probability that a match ofsuspect and crime scene sample would occur by chance. A similarcomparison of markers can be used to identify an individual's remains.For example the U.S. armed forces collect and archive a tissue samplefor each service member. If unidentified human remains are suspected tobe those of an individual a sample from the remains can be analyzed formarkers and compared to the markers present in the tissue sampleinitially collected from that individual.

Marker Assisted Breeding

Genetic markers can assist breeders in the understanding, selecting andmanaging of the genetic complexity of animals and plants. Agricultureindustry, for example, has a great deal of incentive to try to producecrops with desirable traits (high yield, disease resistance, taste,smell, color, texture, etc.) as consumer demand increases andexpectations change. However, many traits, even when the molecularmechanisms are known, are too difficult or costly to monitor duringproduction. Readily detectable polymorphisms which are in close physicalproximity to the desired genes can be used as a proxy to determinewhether the desired trait is present or not in a particular organism.This provides for an efficient screening tool which can accelerate theselective breeding process.

EXAMPLES Example 1

Digestion: Digest 300 ng human genomic in a 20 μl reaction in 1×NEBbuffer 2 with 1×BSA and 1 U/μl Xba1 (NEB). Incubate the reaction at 37°C. overnight or for 16 hours. Heat inactivate the enzyme at 70° C. for20 minutes.

Ligation: Mix the 20 μl digested DNA with 1.25 μl of 5 μM adaptor, 2.5μl 10× ligation buffer and 1.25 μl 400 U/μl ligase. The finalconcentrations are 12 ng/μl DNA, 0.25 μM adaptor, 1× buffer and 20 U/μlligase. Incubate at 16° C. overnight. Heat inactivate enzyme at 70° C.for 20 minutes. Sample may be stored at −20° C.

Amplification: Mix the 25 μl ligation reaction in a 1000 ul PCRreaction. Final concentrations of reagents are as follows: 1×PCR buffer,250 μM dNTPs, 2.5 mM MgCl₂, 0.5 μM primer, 0.3 ng/μl ligated DNA, and0.1 U/μl Taq Gold. The reaction is divided into 10 tubes of 100 μl eachprior to PCR.

Reaction cycles are as follows: 95° C. for 10 minutes; 20 cycles of 95°for 20 seconds, 58° C. for 15 seconds and 72° C. for 15 seconds; and 25cycles of 95° C. for 20 seconds, 55° C. for 15 seconds, and 72° C. for15 seconds followed by an incubation at 72° C. for 7 minutes and thenincubation at 4° C. indefinitely. Following amplification 3 μl of thesample may be run on a 2% TBE minigel at 100V for 1 hour.

Fragmentation and Labeling: PCR reactions were cleaned and concentratedusing a Qiagen PCR clean up kit according to the manufacturer'sinstructions. Eluates were combined to obtain a sample withapproximately 20 μg DNA, approximately 250-300 μl of the PCR reactionwas used. The 20 μg product should be in a volume of 43 μl, if necessaryvacuum concentration may be required. The DNA in 43 μl was combined with5 μl 10×NEB buffer 4, and 2 μl 0.09 U/μl DNase and incubated at 37° C.for 30 min, 95° C. for 10 minutes then to 4° C. DNA was labeled with TdTunder standard conditions.

Hybridization: Standard procedures were used for hybridization, washing,scanning and data analysis. Hybridization was to an array designed todetect the presence or absence of a collection of human SNPs present onXbaI fragments of 400 to 1,000 base pairs.

Example 2

Genomic DNA was digested with XbaI by mixing 5 μl 50 ng/μl human genomicDNA (Coriell Cell Repositories) with 10.5 μl H₂O (Accugene), 2 μl 10×REbuffer 2 (NEB, Beverly, Mass.), 2 μl 10×BSA (NEB, Beverly, Mass.), and0.5 μl XbaI (NEB, Beverly, Mass.). The reaction was incubated at 30° C.for 2 hours, then the enzyme was inactivated by incubation at 70° C. for20 min and then to 4° C. The reaction may be stored at −20° C.

For ligation of the adapters the digested DNA was then mixed with 1.25μl 5 uM adaptor in TE pH 8.0, 2.5 μl T4 DNA ligation buffer and 1.25 μlT4 DNA Ligase (NEB, Beverly, Mass.) which is added last. The reactionwas incubated at 16° C. for 2 hours then at 70° C. for 20 min and thento 4° C. The 25 μl ligation mixture is then diluted with 75 μl H₂O andmay be stored at −20° C.

For PCR 10 μl of the diluted ligated DNA is mixed with 10 μl PCR bufferII (Perkin Elmer, Boston, Mass.), 10 μl 2.5 mM dNTP (PanVera Takara,Madison, Wis.), 10 μl 25 mM MgCl₂, 7.5 μl 10 μM primer (for a finalconcentration of 0.75 μM), 2 μl 5 U/μl Taq Gold (Perkin Elmer, Boston,Mass.) and 50.5 μl H₂O. For each array four 100 μl reactions wereprepared. Amplification was done using the following program: 95° C. for3 min; 35 cycles of 95° C. for 20 sec, 59° C. for 15 sec and 72° C. for15 sec; and a final incubation at 72° C. for 7 min. The reactions werethen held at 4° C. The lid heating option was selected.

The PCR reactions were then purified by mixing the 100 μl PCR reactionwith 500 μl PB or PM buffer into Qiagen columns (Valencia, Calif.) andthe column was centrifuged at 13,000 rpm for 1 min. Flow through wasdiscarded and 750 μl PE buffer with ethanol was added into the column towash the sample and the column was spun at 13,000 rpm for 1 min. Theflow through was discarded and the column was spun at 13,000 rpm foranother 1 min. The flow through was discarded and the column was placedin a new collection tube. For 2 of the 4 samples 30 μl of EB elutionbuffer pH 8.5 was added to the center of the QIAquick membrane to elutethe sample and the columns were allowed to stand at room temperature for5 min and then centrifuged at 13,000 for 1 min. The elution buffer fromthe first 2 samples was then used to elute the other 2 samples and theeluates were combined. The DNA was quantified and diluted so that 48 μlcontains 20 μg DNA.

The DNA was fragmented by mixing 48 μl DNA (20 μg), 5 μl RE Buffer 4,and 2 μl 0.09 U/μl DNase in a total volume of 55 μl. The reaction wasincubated at 37° C. for 30 min then 95° C. for 15 min and then held at4° C.

Fragments were labeled by incubating 50 μl fragmented DNA, 13 μl 5×TdTbuffer (Promega, Madison, Wis.), 1 μl 1 mM biotinolated-ddATP (NEN LifeSciences, Boston, Mass.), and 1 p TdT (Promega, Madison, Wis.) at 37° C.overnight then at 95° C. for 10 min, then held at 4° C.

Hybridization mix is 12 μl 1.22 M MES, 13 μl DMSO, 13 μl 50×Denharts, 3μl 0.5M EDTA, 3 μl 10 mg/mi herring sperm DNA, 3 μl 10 nM oligo B2, 3 μl1 mg/ml Human Cot-1, 3 μl 1% Tween-20, and 140 μl 5M TMACL. 70 μllabeled DNA was mixed with 190 μl hybridization mix. The mixture wasincubated at 95° C. for 10 min, spun briefly and held at 47.5° C. 200 μlof the denatured mixture was hybridized to an array at 47.5° C. for 16to 18 hours at 60 rpm.

Staining mix was 990 μl H₂O, 450 μl 20×SSPE, 15 μl Tween-20, 30 μl 50%Denharts. For the first stain mix 495 μl staining mix with 5 μl 1 mg/mlstreptavidin (Pierce Scientific, Rockford, Ill.), for the second stainmix 495 μl staining mix with 5 μl 0.5 mg/ml biotinylatedanti-streptavidin antibody (Vector Labs, Burlingame, Calif.) and for thethird stain mix 495 μl staining mix with 5 μl 1 mg/ml streptavidin,R-phycoerythrin conjugate (Molecular Probes, Eugene, Oreg.). Wash andstain under standard conditions.

CONCLUSION

From the foregoing it can be seen that the present invention provides aflexible and scalable method for analyzing complex samples of DNA, suchas genomic DNA. These methods are not limited to any particular type ofnucleic acid sample: plant, bacterial, animal (including human) totalgenome DNA, RNA, cDNA and the like may be analyzed using some or all ofthe methods disclosed in this invention. This invention provides apowerful tool for analysis of complex nucleic acid samples. Fromexperiment design to isolation of desired fragments and hybridization toan appropriate array, the above invention provides for fast, efficientand inexpensive methods of complex nucleic acid analysis.

All publications and patent applications cited above are incorporated byreference in their entirety for all purposes to the same extent as ifeach individual publication or patent application were specifically andindividually indicated to be so incorporated by reference. Although thepresent invention has been described in some detail by way ofillustration and example for purposes of clarity and understanding, itwill be apparent that certain changes and modifications may be practicedwithin the scope of the appended claims.

1-33. (canceled)
 34. A method for selecting a collection of targetsequences comprising: identifying fragments that are in a selected sizerange when a genome is digested with a selected enzyme or enzymecombination; identifying sequences of interest present on the fragmentsin the selected size range; and selecting as target sequences: fragmentsthat are in the selected size range and comprise a sequence of interest.35. A collection of target sequences selected according to the method ofclaim
 34. 36-40. (canceled)
 41. The method of claim 34 wherein saidsequences of interest comprise sequence variations. 42-68. (canceled)69. A method for screening for DNA sequence variations in a populationof individuals comprising: providing a first nucleic acid sample fromeach of said individuals; providing a second nucleic acid sample by amethod comprising: fragmenting said first nucleic acid sample to producefragments; ligating at least one adaptor sequence to the population offragments; and, generating a second nucleic acid sample from said firstnucleic acid sample wherein said second nucleic acid sample is enrichedfor a subset of fragments and said subset of fragments comprisessequences from said collection of target sequences; providing aplurality of nucleic acid arrays wherein said arrays comprise probesdesigned to interrogate for DNA sequence variations; hybridizing each ofsaid second nucleic acid samples to one of said plurality of arrays;generating a plurality of hybridization patterns resulting from saidhybridizations; and analyzing the hybridization patterns to determinethe presence or absence of sequence variation in the population ofindividuals.
 70. The method of claim 69 wherein said sequence variationis one or more single nucleotide polymorphisms.
 71. The method of claim70 wherein at least one of the single nucleotide polymorphisms isassociated with a phenotype.
 72. The method of claim 70 wherein at leastone of the single nucleotide polymorphisms is associated with a disease.73. The method of claim 70 wherein at least one of the single nucleotidepolymorphisms is associated with the efficacy of a drug.
 74. The methodof claim 70 wherein at least one of the single nucleotide polymorphismsis associated with a haplotype.
 75. The method of claim 69 wherein thenucleic acid array is designed to interrogate sequence variations insaid collection of target sequences. 76-77. (canceled)
 78. A method foranalyzing a first nucleic acid sample comprising: obtaining a secondnucleic acid sample that is enriched for a subset of fragments saidsubset comprising selected target sequences by a method comprising:selecting one or more target sequences that may be present in the firstnucleic acid sample; fragmenting the first nucleic acid sample so thatthe selected target sequences are present on fragments of a specificsize range; ligating at least one adaptor sequence to the fragments;generating said second nucleic acid sample by amplifying the fragmentsso that the fragments containing the selected target sequences areenriched in the amplified product; providing a nucleic acid array;hybridizing said second nucleic acid sample to said array; and analyzinga hybridization pattern resulting from said hybridization.
 79. Themethod of claim 78 wherein said second nucleic acid sample comprises atleast 0.01% of said first nucleic acid sample. 80-89. (canceled)
 90. Themethod of claim 78 wherein said method for analyzing a first nucleicacid sample comprises determining whether the first nucleic acid samplecontains sequence variations.
 91. The method of claim 90 wherein saidsequence variations are single nucleotide polymorphisms.
 92. The methodof claim 91 wherein at least one of the single nucleotide polymorphismsis associated with a phenotype.
 93. The method of claim 91 wherein atleast one of the single nucleotide polymorphisms is associated with adisease.
 94. The method of claim 91 wherein at least one of the singlenucleotide polymorphisms is associated with the efficacy of a drug. 95.The method of claim 91 wherein at least one of the single nucleotidepolymorphisms is associated with a haplotype.
 96. The method of claim 78wherein said target sequences are selected by a method comprising:identifying fragments that are in a selected size range when a genome isdigested with a selected enzyme or enzyme combination; identifyingsequences of interest present on the fragments in the selected sizerange; and selecting as target sequences fragments that are in theselected size range and comprise a sequence of interest. 97-98.(canceled)